?-agonists, a first line asthma drug, target the beta-2-adrenergic receptors (?2ARs) on airway smooth muscle (ASM) to inhibit the ASM contraction that causes airway narrowing and wheezing. For almost 40 years, the limitations of ?-agonist therapy has been a hotly debated topic. Our work since the inception of this grant has explored multiple modes of GPCR regulation in multiple airway cells, and has emphasized the role of ?2AR desensitization as an important mechanism that limits the therapeutic efficacy of ?-agonists. We have now discovered that the benefits of blocking GRK-mediated ?2AR desensitization in ASM are limited by additional desensitization mechanisms invoked by the second messenger kinase PKA. Because a long term goal of this grant to develop means to thwart ?2AR desensitization and improve the efficacy of ?-agonists as asthma drugs, a significant role of PKA as a feedback regulator of ASM ?2AR presents a profound challenge, given PKA is also presumed to mediate the bronchorelaxant effect of ?-agonists. Studies proposed herein seek to: (Aim 1) identify PKA-dependent mechanisms of ?2AR desensitization and their cooperativity with GRKs; (Aim 2) resolve the ongoing debate as to whether PKA, as opposed to the cAMP effector Epac, is the effector of ?2AR- mediated ASM relaxation, while establishing the PKA-dependence of specific mechanisms of contraction inhibition; and (Aim 3) determine the mechanisms by which compartmentalized cAMP signaling occurs and mediates the anti-contractile effects of ?-agonists in ASM. Experiments will focus on human ASM cell- and tissue- based models of ASM signaling and contraction, and utilize diverse molecular approaches and analytic tools. We anticipate that findings from the proposed studies will provide new insight into the depth and complexity of mechanisms that mediate agonist-specific ?2AR desensitization in a physiological cell. Moreover, we will for the first time in ASM characterize compartmentalized cAMP/PKA signaling and the means to manipulate it, thereby enabling selective enhancement of PKA functions important to the therapeutic effect of ?-agonists.